JP2005257313A - Fluorescence reader - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence reader capable of reading the whole surface of a flat sample at high speed in a short time. <P>SOLUTION: A CCD line sensor 17 is used as a light detection element, and one detection line 32 is simultaneously detected. To simultaneously illuminate the detection line 32 with excitation lights, line forming lasers 11a and 11b emitting sheet-like laser beams 31 and 31b are used. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a fluorescence reading apparatus that reads fluorescence generated from a sample at high speed, and more particularly to a fluorescence reading apparatus suitable for use in reading a biochip.

  A biochip is obtained by immobilizing a biomolecule such as DNA or protein on a substrate such as a glass plate (sometimes called a DNA chip). Conventional fluorescence readers that read fluorescence signals from biochips are roughly divided into two reading methods. One is a reading method using a two-dimensional CCD element, and the other is a reading method using a photomultiplier tube (PMT) by laser scanning. Fluorescence readers using two-dimensional CCD elements are capable of high-speed and high-sensitivity detection due to recent improvements in CCD elements. To read the entire biochip, the biochip is divided into multiple images. It was necessary to detect. At this time, there are sensitivity differences and pixel shifts for each read image, and sensitivity nonuniformity and pixel shifts occur when images are combined. For example, when reading the entire surface of 25 × 75 mm, it is necessary to read the image by dividing it into 25 × 25 mm, and it is necessary to correct the sensitivity unevenness and the pixel shift between the three images. The fluorescence reading apparatus using the PMT by laser scanning can detect with high sensitivity, but it is necessary to scan the entire reading range by laser, for example, about 5 minutes when reading the entire surface of 25 × 75 mm with a resolution of 10 μm, It took about 2 minutes when the resolution was 30 μm.

JP 2000-343254 A

  In the analysis of a biochip, high throughput is important, and it is necessary to shorten the hybridization reaction and the analysis process. Naturally, the reading apparatus is also required to increase the reading speed. However, with the conventional fluorescence reader, it is difficult to read the entire biochip at high speed.

  An object of the present invention is to provide a fluorescence reading apparatus capable of reading the entire surface of a planar sample such as a biochip at high speed in a short time.

  In the present invention, a CCD line sensor is used as the light detection element in order to solve the above problems. In the conventional laser scanning method, it is necessary to scan the entire surface of the reading range (for example, 25 × 75 mm), but by using a CCD line sensor, it is possible to detect one detection line at the same time, and the X axis The entire reading range can be read only by scanning the line in the direction. Further, at this time, it is necessary to simultaneously irradiate the detection line with the excitation light source, but in the present invention, the detection line is simultaneously irradiated with the laser by using a line forming laser that irradiates the sheet-like laser light.

  A fluorescence reading apparatus according to the present invention includes a sample stage that holds a sample, a stage drive unit that drives the sample stage by a distance corresponding to one detection line in a direction crossing the detection line, and a detection line for the sample held on the sample stage. A first line laser that irradiates a sheet-shaped laser beam having a first wavelength on the second line laser; a second line laser that irradiates a sheet-shaped laser beam having a second wavelength different from the first wavelength on the detection line; A first fluorescent filter that transmits fluorescence emitted from the sample by irradiation with laser light of the first wavelength; a second fluorescent filter that transmits fluorescence emitted from the sample by irradiation of laser light of the second wavelength; A CCD line sensor, a light receiving optical system for condensing the fluorescence transmitted through the fluorescence filter to the CCD line sensor, a stage drive unit, a first line laser, and a second line laser. And a Gosuru controller.

  The fluorescence reading apparatus of the present invention can include a fluorescence filter driving unit that drives the first fluorescence filter and the second fluorescence filter. In this case, the control unit includes the first line laser and the second line laser. The first fluorescent filter is positioned on the light receiving optical path when the first line laser is irradiated, and the second fluorescent filter is irradiated when the second line laser is irradiated. Controls the fluorescent filter driving section so as to be positioned on the light receiving optical path of the CCD line sensor.

  The fluorescence reading apparatus of the present invention further includes an optical path switching unit that switches the fluorescence emitted from the sample to an optical path that enters the light receiving lens through the first fluorescent filter and an optical path that enters the light receiving lens through the second fluorescent filter. In this case, the control unit controls the first line laser and the second line laser to irradiate alternately, and the fluorescence from the sample is emitted when the first line laser is radiated. The light path switching unit is controlled so that the fluorescence from the sample enters the light receiving lens through the second fluorescent filter when the second line laser is irradiated through the first fluorescent filter. .

  The fluorescence reading apparatus of the present invention also has a first CCD line sensor that detects fluorescence transmitted through the first fluorescence filter as a CCD line sensor, and a second that detects fluorescence transmitted through the second fluorescence filter. A CCD line sensor, and in that case, as the light receiving optical system, the light that passes through the first fluorescent filter and the light that passes through the second fluorescent filter and the light receiving optical system that collects the fluorescence that has passed through the first fluorescent filter. And a light receiving optical system for condensing the light onto the second CCD line sensor, and the control unit controls the first line laser and the second line laser so that both are irradiated simultaneously.

  According to the present invention, reading can be performed at a higher speed than the conventional fluorescence reading apparatus, and the reading time can be shortened.

  Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Here, a case where fluorescence from a biochip is read will be described. However, a fluorescence reading sample is not necessarily a biochip.

  FIG. 1 is a schematic diagram illustrating a configuration example of a fluorescence reading apparatus according to the present invention. In the present invention, a line laser that generates sheet-like excitation light is used as a light source, and a CCD line sensor is used as a detector. The illustrated apparatus is equipped with two types of lasers, a line laser 10a and a line laser 10b, as excitation light sources. This is because a biological substance such as DNA hybridized with a probe on a biochip is generally labeled with two types of fluorescence, Cy3 and Cy5. In this example, a line laser 10a having a wavelength of 532 nm and an output of 10 mW was used for excitation of Cy3, and a line laser 10b of 635 nm and an output of 10 mW was used for excitation of Cy5. However, depending on the sample, the biological material may be labeled with fluorescence other than Cy3 and Cy5. In this case, detection is possible by mounting a line laser that generates wavelengths other than 532 nm and 635 nm. The line laser 10a is controlled ON / OFF and laser output by the control PC 12 via the laser driver 11a. Similarly, the line laser 10b is controlled by the control PC 12 via the laser driver 11b.

  The sheet-like excitation light generated from the line lasers 10a and 10b irradiates the biochip 22 held on the sample stage 21 linearly. Fluorescence generated from the biochip 22 by the excitation light irradiation passes through the fluorescence filter 13 and is collected on the CCD line sensor 17 by the light receiving lens 16. The fluorescent filter 13 is driven as described later by a fluorescent filter driving motor 15 controlled by a control PC via a fluorescent filter driving motor driver 14. The output signal of the CCD line sensor 17 is stored in the data storage unit 19 via the CCD control unit 18 of the control PC. The sample stage 21 is driven by a stage drive motor 24 controlled by a control PC via a stage drive motor driver 23.

  Details of the excitation optical system will be described with reference to FIG. Both the sheet-like excitation light 31a emitted from the line laser 10a and the sheet-like excitation light 31b emitted from the line laser 10b form the detection line 32 on the biochip 22 in a line shape having a width of 30 μm and a length of 25 mm, for example. Irradiate. The detection line is a range in which reading is performed by one data sampling. The reason why the irradiation range of the excitation light source is the same as that of the detection line in this apparatus is due to two reasons for reducing noise from other than the detection line 32 and preventing image blurring and improving the plane resolution. The line lasers 10a and 10b are arranged so that the incident angles with respect to the biochip 22 are the same in order to make the reading conditions the same. In the illustrated example, the line lasers 10a and 10b are excited at an incident angle of about 70 degrees. Irradiate light. However, the angle of the laser arrangement is not limited to this, and it is preferable that the laser is arranged close to the components of the light receiving optical system and as perpendicular to the biochip as possible. This is to obtain a uniform light distribution within a detection line width of 30 μm.

  FIG. 3 is an explanatory diagram of a configuration example of a line laser. The line laser 10 includes a laser oscillator 32 and a line shaping optical system 33. Various lasers can be used for the laser oscillator 32 regardless of whether they are solid, liquid, or semiconductor. Here, a small semiconductor laser is used in order to reduce the external size of the apparatus. The laser emitted from the laser oscillator 32 enters the line shaping optical system 33 as coherent light. The line shaping optical system 33 is composed of a plurality of lenses, and forms coherent light into a sheet-like laser light 34. The line shaping optical system 33 can be designed exclusively according to the wavelength, line width, and length of the laser. In this embodiment, the line shaping optical system 33 is designed to have a width of 30 μm and a length of 25 mm.

  Returning to FIG. 1, the fluorescence generated from the biological material on the fluorescently labeled biochip is received by the light receiving lens 16 and guided to the CCD line sensor 17. The fluorescence guided to the CCD line sensor 17 is converted into an electric signal by the CCD element of the CCD line sensor 17. At this time, due to the characteristics of the CCD element, the conversion efficiency into the electric signal differs depending on the exposure time of the fluorescence to the CCD element. If the exposure time is long, a small amount of fluorescence is converted into a large electrical signal, and fluorescence detection with high sensitivity is possible. However, noise is amplified accordingly, and the reading time is also long. In this apparatus, the exposure time can be controlled from the control PC 12 via the CCD control unit 18, and high sensitivity detection by long exposure or low sensitivity detection by short reading can be selected. In this embodiment, the exposure time can be set in units of 1 ms from 1 ms to 1 second.

  The light receiving optical system will be described with reference to FIG. FIG. 4 is a side view of the light receiving optical system as seen from the side. The fluorescence from the detection line on the biochip 22 excited by the line laser is received by the light receiving lens 16 through the fluorescent filter 13 and condensed on the CCD line sensor 17. In this embodiment, a fluorescent filter that transmits a wavelength range of 570 nm ± 20 nm is used as a fluorescent filter for Cy3, two fluorescent filters that transmit a wavelength range of 670 nm ± 20 nm are used as a fluorescent filter for Cy5, and 8 is used as a CCD line sensor 17. A CCD line sensor with 1,000 pixels was adopted. By adopting a CCD line sensor of 8,000 pixels, reading with a pixel resolution of about 30 μm becomes possible. The pixel resolution in this case is the actual size on the biochip represented by one pixel.

  In this case, as shown in FIG. 5, a line having a width of 30 μm can be detected with a pixel resolution of about 30 μm × 30 μm, and is sufficient for measuring a sample diameter of about 200 μm on a general biochip. Considered resolution. In addition, it is possible to change the pixel resolution by changing the irradiation width of the excitation light source, the aperture of the light receiving lens, and the number of pixels of the CCD line sensor camera. System and CCD line sensor camera need to be selected.

  Next, the configuration of the fluorescent filter will be described with reference to FIG. The fluorescent filter 13 includes fluorescent filters 13a and 13b. The fluorescent filter 13a is for detecting Cy3 and is used as a pair with the line laser 10a. The fluorescent filter 13b is for detecting Cy5 and is used as a pair with the line laser 10b. The fluorescent filter 13 is connected to the fluorescent filter drive motor 15 and can be controlled from the control PC 12 via the fluorescent filter drive motor driver 14. The control PC 12 controls the fluorescent filter 13a so as to be positioned in front of the light receiving lens 16 at the same time when the line laser 10a is emitted, and to position the fluorescent filter 13b in front of the light receiving lens 16 when the line laser 10b is emitted. The

  FIG. 7 is a schematic diagram illustrating another example of a mechanism for switching a fluorescent filter. This fluorescence filter switching mechanism allows the CCD line sensor 17 to detect fluorescence from Cy3 and fluorescence from Cy5 in a time-division manner by switching the optical path without mechanically driving the two fluorescence filters 13a and 13b. It is a thing.

  The fluorescence filter switching mechanism of the present example includes two reflection mirrors 41 and 42 disposed with the Cy3 detection fluorescence filter 13a interposed therebetween, and two reflection mirrors 43 and 44 disposed with the Cy5 detection fluorescence filter 13b interposed therebetween. , And two galvanometer mirrors 45 and 46 and a galvanometer mirror driving unit 47. The galvanometer mirror driving unit 47 receives the control signal from the control PC 12 and drives the two galvanometer mirrors 45 and 46 simultaneously. When the line laser 10a emits light, the two galvanometer mirrors 45 and 46 are set at the angular positions indicated by the solid lines. At this time, the fluorescence generated from the biochip 22 is received by the light receiving lens 16 through the fluorescent filter 13 a and guided to the CCD line sensor 17. On the other hand, when the line laser 10b emits light, the two galvanometer mirrors 45 and 46 are set at the angular positions indicated by dotted lines, and the fluorescence generated from the biochip passes through the fluorescence filter 13b and passes through the light receiving lens 16. Light is received and guided to the CCD line sensor 17. In this way, the control PC 12 drives the two line lasers 10a and 10b and the two galvanometer mirrors 45 and 46 in synchronization, thereby causing the fluorescence emitted from Cy3 and the fluorescence emitted from Cy5 in the sample. Can be detected with temporal separation. When this fluorescent filter switching mechanism is used, the two fluorescent filters 13a and 13b can be switched at higher speed.

  Returning to FIG. 1, the fluorescence is converted into an electrical signal by the CCD line sensor 17, transferred as data for each detection line 32 to the CCD control unit 18 in the control PC, and stored as data on the control PC. At this time, since data is transferred from the CCD line sensor 17 for each line detection, all the data is stored in the data storage unit 19 on the control PC. At the same time, the control PC controls the stage drive motor 24 via the stage drive motor driver 23, moves the sample stage 21 by one line, and moves to detection of the next detection line. The amount of movement of the sample stage by the stage drive motor 24 is 30 μm according to the pixel resolution.

  The flow of the reading process at this time is summarized in FIG. In starting reading, first, reading conditions (reading range, exposure time) are input (S11). Next, the control PC starts laser emission, and at the same time, selects a fluorescent filter in accordance with the laser emission (S12). Thereafter, the stage drive motor 24 is controlled to move the sample stage 21 to the reading start position (S13). Thereafter, the CCD line sensor camera is exposed for a time set by the user (S14). After the exposure is completed, data is transferred from the CCD line sensor camera to the data storage unit in the control PC (S15). Thereafter, the sample stage 21 is moved by one line by the stage moving motor control 24 (S16). At this time, if reading of the entire reading range is not completed (S17), exposure, data transfer, and line movement of the CCD line sensor camera are performed again, and this is repeated until reading of the entire reading range is completed. The data for each line during this time is stored in the data storage unit 19 of the control PC 12. When the reading of the entire reading range is finished, the laser emission is finished (S18), and the reading is finished. At this time, when reading two wavelengths sequentially, the process from the start of reading to the end of reading is performed twice for each laser to be emitted, and when reading two wavelengths at the same time, two lasers are emitted at the same time, so one process is performed. It becomes.

  After the reading is completed, the data for each line stored in the data storage unit is combined and output as two-dimensional image data. At this time, the reading time per detection line was set to 10 milliseconds. As a result, the entire biochip area of 25 × 75 mm can be read in 25 seconds. In addition, when the detection time per detection line is 1 msec in the low sensitivity mode, reading in 2.5 seconds is possible.

  FIG. 9 is a diagram illustrating an example of a light receiving optical system in the case of two-wavelength simultaneous reading. The line lasers 10a and 10b are emitted simultaneously, and the fluorescent reagents Cy3 and Cy5 on the biochip 22 emit fluorescence simultaneously. At this time, the fluorescence generated by Cy3 is detected by the fluorescence filter 13a paired with the line laser 10a, the light receiving lens 16a and the CCD line sensor 17a, and the fluorescence generated by Cy5 is detected by the fluorescence filter 13b paired with the line laser 10b. It is detected by the lens 16b and the CCD line sensor 17b.

1 is a schematic diagram showing a configuration example of a fluorescence reading apparatus according to the present invention. The figure explaining an excitation optical system. The figure explaining the structural example of a line laser. The figure explaining a light-receiving optical system. The figure explaining the laser irradiation width and pixel resolution on a biochip. The figure explaining the structural example of a fluorescence filter. Schematic which shows the other example of a fluorescence filter switching mechanism. The figure explaining the flow of a reading process. Schematic of the light receiving optical system in the case of two-wavelength simultaneous reading.

Explanation of symbols

10a, 10b ... line laser, 11a, 11b ... laser driver, 12 ... control PC, 13 ... fluorescent filter, 14 ... fluorescent filter drive motor driver, 15 ... fluorescent filter drive motor, 16 ... light receiving lens, 17 ... CCD line sensor , 18 ... CCD control unit, 19 ... data storage unit, 21 ... sample stage, 22 ... biochip, 23 ... stage drive motor driver, 24 ... stage drive motor, 31a, 31b ... sheet-like excitation light, 32 ... laser oscillator , 33 ... line shaping optical system, 34 ... sheet-like laser light, 41 to 44 ... reflection mirror, 45 and 46 ... galvano mirror, 47 ... galvano mirror drive unit

Claims (4)

A sample stage for holding the sample;
A stage drive unit for driving the sample stage in a direction intersecting the detection line by a distance corresponding to one detection line;
A first line laser that irradiates a sheet-like laser beam having a first wavelength onto a sample detection line held on the sample stage;
A second line laser that irradiates a sheet-like laser beam having a second wavelength different from the first wavelength onto the detection line;
A first fluorescent filter that transmits the fluorescence emitted from the sample by irradiation with the laser beam of the first wavelength;
A second fluorescent filter that transmits fluorescence emitted from the sample by irradiation with the laser beam of the second wavelength;
A CCD line sensor,
A light receiving optical system for condensing the fluorescence transmitted through the fluorescent filter onto the CCD line sensor;
A fluorescence reading apparatus comprising: the stage driving unit; and a control unit that controls the first line laser and the second line laser.   2. The fluorescence reading apparatus according to claim 1, further comprising a fluorescence filter driving unit that drives the first fluorescence filter and the second fluorescence filter, wherein the control unit includes the first line laser and the second line. The laser is controlled so as to irradiate alternately, and when the first line laser irradiates, the first fluorescent filter is positioned on the light receiving optical path, and when the second line laser irradiates The fluorescence reading apparatus, wherein the fluorescence filter driving unit is controlled so that the second fluorescence filter is positioned on a light receiving optical path of the CCD line sensor.   2. The fluorescent light reading apparatus according to claim 1, wherein fluorescence emitted from a sample is switched between an optical path for entering the light receiving lens through the first fluorescent filter and an optical path for entering the light receiving lens through the second fluorescent filter. A switching unit, and the control unit controls to irradiate the first line laser and the second line laser alternately, and from the sample when the first line laser is irradiating Fluorescence enters the light receiving lens through the first fluorescent filter, and fluorescence from the sample enters the light receiving lens through the second fluorescent filter when the second line laser is irradiated. As described above, the fluorescence reading apparatus is characterized by controlling the optical path switching unit.   2. The fluorescence reader according to claim 1, wherein the CCD line sensor detects a first CCD line sensor that detects fluorescence that has passed through the first fluorescence filter, and fluorescence that has passed through the second fluorescence filter. A second CCD line sensor, and a light receiving optical system for condensing the fluorescence transmitted through the first fluorescent filter as the light receiving optical system on the first CCD line sensor and the second fluorescent filter. And a light receiving optical system for condensing the fluorescent light on the second CCD line sensor, and the control unit controls the first line laser and the second line laser so that both are irradiated simultaneously. A fluorescence reading apparatus.

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